In Situ Detection of ROS

H₂O₂ and superoxide anions (O²⁻) were detected in situ using 3,3ʹ-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining as previously described (Ren et al., 2019 (link)). Briefly, WT and dek66 mutant kernels isolated from self-pollinated segregating ears at 15 DAP were longitudinally cut and immersed in 1 mg ml⁻¹ DAB (Sigma-Aldrich) in 50 mM Tris–HCl buffer (pH 5.0) at room temperature in dark for 12 h to detect hydrogen peroxide (H₂O₂). NBT staining was performed to detect O²⁻. Briefly, the kernels were immersed in 0.5 mg ml⁻¹ NBT solution in 10 mM potassium phosphate buffer (pH 7.6) and incubated in complete darkness at room temperature for 20 min. The images were taken using an Olympus DP72 microscope.